PROJECT SUMMARY Specialization of Innate and Adaptive Immune Cells in Intestinal Barrier Function. Maintenance of mucosal barrier integrity requires the interplay of innate and adaptive immune cells with the mucosal epithelium. Interleukin-22 (IL-22) is a cytokine of the IL-10 family that is produced by type 3 immune cells, such as group 3 innate lymphoid cells (ILC3s) and cells of the Th17 pathway, and acts on epithelial cells of barrier tissues to prevent invasion of certain microbes, particularly extracellular bacteria and fungi. The basis for the protective actions of IL-22 on intestinal epithelial cells (IECs) is not fully understood, but it enhances their production of antimicrobial factors, maintains epithelial tight junctions and promotes the proliferation and survival of IECs. Like many immune cytokines that participate in host defense, IL-22 is upregulated in chronic immune-mediated diseases, and it appears to play a protective role in inflammatory bowel disease (IBD), perhaps by restraining the epithelial damage caused by dysregulated T cell responses against constituents of the intestinal microbiome. However, the hyperproliferative effects of IL-22 have also been implicated in epithelial malignant transformation that leads to colorectal cancer (CRC). We and others have shown that during infectious colitis modeled by the enteropathogen, C. rodentium, two phases of IL-22 production can be distinguished: an early phase dominated by IL-22?producing innate immune cells, and a late phase that is dominated by IL-22? producing CD4 T cells. While both innate and adaptive immune cells produce IL-22 during infection, their relative contributions to host protection are unknown, as are details of the mechanisms by which IL-22 acts. In preliminary studies, we have generated novel IL-22 reporter/conditional knockout (cKO) mice with which to track and/or delete specific subsets of IL-22-producing immune cells. Remarkably, we find that the locations and functions of IL-22?producing cells during C. rodentium infection are distinct: innate immune cells, dominated by ILC3s, localize primarily to isolated lymphoid follicles and activate superficial IECs at initial sites of bacterial colonization. However, ILC3s fail to protect the intestinal crypts, which are invaded by bacteria in mice with IL-22 deficiency targeted to T cells. Thus, IL-22?producing T cells appear to be indispensible for protection of the intestinal crypts via their activation of crypt-lining epithelium. We hypothesize that IL-22? producing innate cells are limited to rapid activation of superficial IECs at initial sites of bacterial attachment, whereas IL-22?producing T cells are required to prevent bacterial invasion of crypts. Moreover, superficial and crypt IECs differ in their response to IL-22-dependent innate and adaptive signals. In this proposal we will define the relative contributions of innate and adaptive immune cells to IL-22?dependent activation of different subsets of colonic epithelial cells, and define the gene expression programs elicited by IL-22 in the context of innate or adaptive immune cell actions. These studies will reveal new insights into specialization of immune cell subsets in intestinal host defense and mechanisms that control intestinal inflammation in IBD and CRC.